Datasets:

lhallee

/

context_pubmed

like 0

Animal studies show aging varies between individuals as well as between organs within an individual^1-4, but whether this is true in humans and its effect on age-related diseases is unknown. We utilized levels of human blood plasma proteins originating from specific organs to measure organ-specific aging differences in living individuals. Using machine learning models, we analysed aging in 11 major organs and estimated organ age reproducibly in five independent cohorts encompassing 5,676 adults across the human lifespan. We discovered nearly 20% of the population show strongly accelerated age in one organ and 1.7% are multi-organ agers. Accelerated organ aging confers 20-50% higher mortality risk, and organ-specific diseases relate to faster aging of those organs. We find individuals with accelerated heart aging have a 250% increased heart failure risk and accelerated brain and vascular aging predict Alzheimer's disease (AD) progression independently from and as strongly as plasma pTau-181 (ref. ⁵), the current best blood-based biomarker for AD. Our models link vascular calcification, extracellular matrix alterations and synaptic protein shedding to early cognitive decline. We introduce a simple and interpretable method to study organ aging using plasma proteomics data, predicting diseases and aging effects.

Fatty acid-binding protein 7 (FABP7) is vital for uptake and trafficking of fatty acids in the nervous system. To investigate the involvement of FABP7 in noise-induced hearing loss (NIHL) pathogenesis, we used Fabp7 knockout (KO) mice generated via CRISPR/Cas9 in the C57BL/6 background. Initial auditory brainstem response (ABR) measurements were conducted at 9 weeks, followed by noise exposure at 10 weeks. Subsequent ABRs were performed 24 h later, with final measurements at 12 weeks. Inner ears were harvested 24 h after noise exposure for RNA sequencing and metabolic analyses. We found no significant differences in initial ABR measurements, but Fabp7 KO mice showed significantly lower thresholds in the final ABR measurements. Hair cell survival was also enhanced in Fabp7 KO mice. RNA sequencing revealed that genes associated with the electron transport chain were upregulated or less impaired in Fabp7 KO mice. Metabolomic analysis revealed various alterations, including decreased glutamate and aspartate in Fabp7 KO mice. In conclusion, FABP7 deficiency mitigates cochlear damage following noise exposure. This protective effect was supported by the changes in gene expression of the electron transport chain, and in several metabolites, including excitotoxic neurotransmitters. Our study highlights the potential therapeutic significance of targeting FABP7 in NIHL.

Fish trypanosomiasis is a common blood parasitic disease transmitted by aquatic invertebrates, such as leeches. This study aims to shed light on the cytotoxicity of Trypanosoma sp. on erythrocytes and its impacts on the innate immune response (serum lysozyme activity, nitric oxide production, phagocytic activity, serum total protein, and globulin) in wild African catfish, Clarias gariepinus. One hundred catfish were examined using blood smears stained with Giemsa and confirmed with PCR. The prevalence of infection was found to be 10% by microscope detection and 15% by PCR. The morphological identification of Trypanosoma as Trypanosoma mukasai was determined. Additionally, this study included previously undescribed features of Trypanosoma, such as the width of the anterior and posterior body, the length of the posterior pale region, and the number of folds. Various alterations in erythrocytes were observed, totaling 54.57%. Nuclear abnormalities, including fragmented nuclei, eccentric nuclei, and micronuclei, were also reported. Infected fish showed a reduction in serum total protein and globulin levels, while nitric oxide production, lysozyme activity, and phagocytic activity exhibited a significant increase compared to non-infected fish. We believe that our findings will contribute valuable data to the morphological and molecular identification of Trypanosoma sp. in African catfish, as well as their cytotoxic impact.

Feather, horn, hoof, and other keratin waste are protein-rich but limited by natural keratinase synthesis, activity, pH, and temperature stability. It is challenging to realize its large-scale application in industries. Bacillus subtilis spores are a safe, efficient, and highly resistant immobilized carrier, which can improve target proteins' resistance. In this research, KERQ7, the keratinase gene of Bacillus tequilensis strain Q7, was fused to the Bacillus subtilis genes coding for the coat proteins CotG and CotB, respectively, and displayed on the surface of B. subtilis spores. Compared with the free KERQ7, the immobilized KERQ7 showed a greater pH tolerance and heat resistance on the spore surface. The activity of CotG-KERQ7 is 1.25 times that of CotB-KERQ7, and CotG-KERQ7 is more stable. When the flexible linker peptide L3 was used to connect CotG and KERQ7, the activity was increased to 131.2 ± 3.4%, and the residual enzyme activity was still 62.5 ± 2.2% after being kept at 60 ℃ for 4 h. These findings indicate that the flexible linker and CotG were more effective for the spore surface display of keratinase to improve stress resistance and promote its wide application in feed, tanning, washing, and other industries.

Environmental pollution is a global concern. Various organic compounds are released into the environment through wastewater, waste gas, and waste residue, ultimately accumulating in the environment and the food chain. This poses a significant threat to both human health and ecology. Currently, a growing body of research has demonstrated that microorganisms employ their Cytochrome P450 (CYP450) system for biodegradation, offering a crucial approach for eliminating these pollutants in environmental remediation. CYP450, a ubiquitous catalyst in nature, includes a vast array of family members distributed widely across various organisms, including bacteria, fungi, and mammals. These enzymes participate in the metabolism of diverse organic compounds. Furthermore, the rapid advancements in enzyme and protein engineering have led to increased utilization of engineered CYP450s in environmental remediation, enhancing their efficiency in pollutant removal. This article presents an overview of the current understanding of various members of the CYP450 superfamily involved in transforming organic pollutants and the engineering of biodegrading CYP450s. Additionally, it explores the catalytic mechanisms, current practical applications of CYP450-based systems, their potential applications, and the prospects in bioremediation.

α-Arbutin, a naturally occurring glycosylated derivative of hydroquinone (HQ), effectively inhibits melanin biosynthesis in epidermal cells. It is widely recognized as a fourth-generation whitening agent within the cosmetic industry. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Sucrose phosphorylase (SPase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. In this study, a previously reported SPase known for its effectiveness in synthesizing α-arbutin, was used as a probe sequence to identify a novel SPase from Paenibacillus elgii (PeSP) in the protein database. The sequence similarity between PeSP and the probe was 39.71%, indicating a degree of novelty. Subsequently, the gene encoding PeSP was coexpressed with the molecular chaperone pG-Tf2 in Escherichia coli, significantly improving PeSP's solubility. Following this, PeSP was characterized and employed for α-arbutin biosynthesis. The specific activity of co-expressed PeSP reached 169.72 U/mg, exhibited optimal activity at 35℃ and pH 7.0, with a half-life of 3.6 h under the condition of 35℃. PeSP demonstrated excellent stability at pH 6.5-8.5 and sensitivity to high concentrations of metal ions. The kinetic parameters K_m and k_cat/K_m were determined to be 14.50 mM and 9.79 min^- 1·mM^- 1, respectively.The reaction conditions for α-arbutin biosynthesis using recombinant PeSP were optimized, resulting in a maximum α-arbutin concentration of 52.60 g/L and a HQ conversion rate of 60.9%. The optimal conditions were achieved at 30℃ and pH 7.0 with 200 U/mL of PeSP, and by combining sucrose and hydroquinone at a molar ratio of 5:1 for a duration of 25 h.

Every novel infection requires an assessment of the host response coupled with identification of unique biomarkers for predicting disease pathogenesis, treatment targets and diagnostic utility. Studies have exposed dysregulated inflammatory response induced by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as significant predictor or cause of disease severity/prognosis and death. This study evaluated inflammatory biomarkers induced by SARS-CoV-2 in plasma of patients with varying disease phenotypes and healthy controls with prognostic or therapeutic potential. We stratified SARS-CoV-2 plasma samples based on disease status (asymptomatic, mild, severe, and healthy controls), as diagnosed by RT-PCR SARS-CoV-2. We used a solid phase sandwich and competitive Enzyme-Linked Immunosorbent Assay (ELISA) to measure levels of panels of immunological (IFN-γ, TNF-α, IL-6, and IL-10) and biochemical markers (Ferritin, Procalcitonin, C-Reactive Protein, Angiotensin II, Homocysteine, and D-dimer). Biomarker levels were compared across SARS-CoV-2 disease stratification. Plasma IFN-γ, TNF-α, IL-6, and IL-10 levels were significantly (P < 0.05) elevated in the severe SARS-CoV-2 patients as compared to mild, asymptomatic, and healthy controls. Ferritin, Homocysteine, and D-dimer plasma levels were significantly elevated in severe cases over asymptomatic and healthy controls. Plasma C-reactive protein and Angiotensin II levels were significantly (P < 0.05) higher in mild than severe cases and healthy controls. Plasma Procalcitonin levels were significantly higher in asymptomatic than in mild, severe cases and healthy controls. Our study demonstrates the role of host inflammatory biomarkers in modulating the pathogenesis of COVID-19. The study proposes a number of potential biomarkers that could be explored as SARS-CoV-2 treatment targets and possible prognostic predictors for a severe outcome. The comprehensive analysis of prognostic biomarkers may contribute to the evidence-based management of COVID-19 patients.

Mounting evidence indicates that the gut microbiota influences the neurodevelopment and behavior of insects through the gut-brain axis. However, it is currently unclear whether the gut microbiota affect the head profiles and immune pathway in pests. Here, we find that gut bacteria is essential for the immune and neural development of adult Spodoptera frugiperda, which is an extremely destructive agricultural pest worldwide. 16 S rRNA sequencing analysis showed that antibiotics exposure significantly disturbed the composition and diversity of gut bacteria. Further transcriptomic analysis revealed that the adult head transcripts were greatly affected by gut dysbacteriosis, and differently expression genes critical for brain and neural development including A4galt, Tret1, nsun4, Galt, Mitofilin, SLC2A3, snk, GABRB3, Oamb and SLC6A1 were substantially repressed. Interestingly, the dysbacteriosis caused sex-specific differences in immune response. The mRNA levels of pll (serine/threonine protein kinase Pelle), PGRP (peptidoglycan-sensing receptor), CECA (cecropin A) and CECB (cecropin B) involved in Toll and Imd signaling pathway were drastically decreased in treated male adults' heads but not in female adults; however, genes of HIVEP2, ZNF131, inducible zinc finger protein 1-like and zinc finger protein 99-like encoding zinc-finger antiviral protein (ZAP) involved in the interferon (IFNα/β) pathway were significantly inhibited in treated female adults' heads. Collectively, these results demonstrate that gut microbiota may regulate head transcription and impact the S. frugiperda adults' heads through the immune pathway in a sex-specific manner. Our finding highlights the relationship between the gut microbiota and head immune systems of S. frugiperda adults, which is an astonishing similarity with the discoveries of other animals. Therefore, this is the basis for further research to understand the interactions between hosts and microorganisms via the gut-brain axis in S. frugiperda and other insects.

Immunotherapy in combination with platinum-etoposide (EP) chemotherapy has been approved as a first-line treatment for extensive-stage small cell lung cancer (ES-SCLC). However, real-world (RW) data regarding the use of immune checkpoint inhibitors (ICIs) in ES-SCLC are lacking. We aimed to assess the differences between programmed death protein 1 (PD-1) inhibitors and programmed death ligand 1 (PD-L1) inhibitors, both in conjunction with EP chemotherapy, as first-line treatment for ES SCLC.

Asthma is a polygenic disease that may onset during childhood. Inhaled corticosteroids (ICS) are the main therapy in asthma, although their efficacy varies among individuals. Nuclear factor κB (NF-κB) is an important target of ICS treatment of asthma. Recent research has reported that GRB2 associated binding protein 1 (GAB1) gene may participate in the pathogenesis of asthma by regulating the NF-κB pathway. Therefore, we used the technique of an improved multiplex ligation detection reaction to sequence GAB1 gene and investigated the involvement of Single-nucleotide variants (SNVs) in GAB1 gene in asthma and ICS efficacy in asthmatic children. We found no differences between asthma cases and controls in allele or genotype frequencies of GAB1. Haplotype analysis showed an increased tendency for AGGAGC frequency in asthma patients compared with controls (OR = 2.69, p = 0.018). The percentage of EOS and genotype distribution of rs1397527 were associated (p = 0.007). The EOS percentage was higher in GT genotype when compared to the GG genotype (5.50 vs 3.00, Bonferroni adjusted p = 0.005). After 12-weeks ICS treatment, GAB1 rs1397527 TT and GT genotype carriers had a smaller change in forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) than GG carriers (p = 0.009), and rs3805236 GG and AG genotype carriers also had a smaller change in FEV1/FVC than AA carriers (p = 0.025). For ICS response, the frequency of GG genotype of rs1397527 was significantly higher in good responders (p = 0.038). The generalized multifactor dimensionality reduction (GMDR) analysis showed a best significant four-order model (rs1397527, allergen exposure, environmental tobacco smoke exposure, and pet exposure) involving gene-environment interactions (p = 0.001). In summary, we found that GAB1 SNVs were not associated with asthma susceptibility. Haplotype AGGAGC was a risk factor for asthma. GAB1 variants were associated with eosinophils and ICS response in asthmatics. Furthermore, gene-environment interaction was observed.

In addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials.

Current effective therapies for autoimmune diseases rely on systemic immunomodulation that broadly affects all T and/or B cell responses. An ideal therapeutic approach would combine autoantigen-specific targeting of both T and B cell effector functions, including efficient removal of pathogenic autoantibodies. Albeit multiple strategies to induce T cell tolerance in an autoantigen-specific manner have been proposed, therapeutic removal of autoantibodies remains a significant challenge. Here, we devised an approach to target both autoantigen-specific T cells and autoantibodies by producing a central nervous system (CNS) autoantigen myelin oligodendrocyte glycoprotein (MOG)-Fc fusion protein. We demonstrate that MOG-Fc fusion protein has significantly higher bioavailability than monomeric MOG and is efficient in clearing anti-MOG autoantibodies from circulation. We also show that MOG-Fc promotes T cell tolerance and protects mice from MOG-induced autoimmune encephalomyelitis. This multipronged targeting approach may be therapeutically advantageous in the treatment of autoimmunity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative and remains incurable. Aluminum is a potent neurotoxin associated with AD. The main pathological features of AD are extracellular amyloid-β protein deposition and intracellular hyperphosphorylated Tau protein. A body of evidence suggest that oxidative stress and autophagy are involved in the pathogenesis of AD. Andrographis paniculata (AP) is a native plant with anti-inflammatory, anti-oxidative stress, and regulation of autophagy properties. AP significantly alleviated cognitive impairments, reduced Aβ deposition and has neuroprotective effect. However, its effects on aluminum-induced AD model have not been studied much. In this study, we investigated whether AP protect against aluminum-induced neurotoxicity through regulation of p62-Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2 related factor 2 (Nrf2) pathway and activation autophagy in vivo and in vitro.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a hypoxic microenvironment, a high rate of heterogeneity as well as a high likelihood of recurrence. Mounting evidence has affirmed that long non-coding RNAs (lncRNAs) participate in the carcinogenesis of PDAC cells. In this study, we revealed significantly decreased expression of GATA6-AS1 in PDAC based on the GEO dataset and our cohorts, and showed that low GATA6-AS1 expression was linked to unfavorable clinicopathologic characteristics as well as a poor prognosis. Gain- and loss-of-function studies demonstrated that GATA6-AS1 suppressed the proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) process of PDAC cells under hypoxia. In vivo data confirm the suppressive roles of GATA6-AS1/SNAI1 in tumor growth and lung metastasis of PDAC. Mechanistically, hypoxia-driven E26 transformation-specific sequence-1 (ETS1), as an upstream modulatory mechanism, was essential for the downregulation of GATA6-AS1 in PDAC cells. GATA6-AS1 inhibited the expression of fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) eraser, and repressed SNAI1 mRNA stability in an m6A-dependent manner. Our data suggested that GATA6-AS1 can inhibit PDAC cell proliferation, invasion, migration, EMT process and metastasis under hypoxia, and disrupting the GATA6-AS1/FTO/SNAI1 axis might be a viable therapeutic approach for refractory hypoxic pancreatic cancers.

Neuroinflammation appears to involve some degree of excitotoxicity promulgated by microglia, which release glutamate via the system x_C^- (Sx_C^-) cystine-glutamate antiporter. With the aim of mitigating this source of neuronal stress and toxicity, we have developed a panel of inhibitors of the Sx_C^- antiporter. The compounds were based on L-tyrosine, as elements of its structure align with those of glutamate, a primary physiological substrate of the Sx_C^- antiporter. In addition to 3,5-dibromotyrosine, ten compounds were synthesized via amidation of that parent molecule with a selection of acyl halides. These agents were tested for the ability to inhibit release of glutamate from microglia activated with lipopolysaccharide (LPS), an activity exhibited by eight of the compounds. To confirm that the compounds were inhibitors of Sx_C^-, two of them were further tested for the ability to inhibit cystine uptake. Finally, these agents were shown to protect primary cortical neurons from the toxicity exhibited by activated microglia. These agents may hold promise in reducing the neurodegenerative effects of neuroinflammation in conditions, such as encephalitis, traumatic brain injury, stroke, or neurodegenerative diseases.

Joint capsule fibrosis, a common complication of joint immobilization, is mainly characterized by abnormal collagen deposition. The present study aimed to investigate the effect of extracorporeal shock wave therapy (ESWT) on reduced collagen deposition in the joint capsule during immobilization-induced joint capsule fibrosis. Additionally, the potential involvement of the adenosine A₂A receptor (A₂AR)-Neurotrophic factor e2-related factor 2 (Nrf2)/Haem oxygenase-1 (HO-1) pathway was explored. Thirty 3-month-old male Sprague-Dawley rats were randomly assigned to five groups: control (C), immobilization model (IM), natural recovery (NR), ESWT intervention (EI), and ESWT combined with A₂AR antagonist SCH 58261 intervention (CI). After the left knee joints of rats in the IM, NR, EI and CI groups were immobilized using a full-extension fixation brace for 4&#160;weeks, the EI and CI groups received ESWT twice a week for 4&#160;weeks. The CI group was also treated with ESWT following intraperitoneal injection of SCH 58261 (0.01&#160;mg/kg) for 4&#160;weeks. The range of motion of the left knee joint was measured, and the protein levels of collagens I and III, A₂AR, phosphorylated-protein kinase A/protein kinase A (p-PKA/PKA), p-Nrf2/Nrf2, and HO-1 were analysed by Western blotting. The IM and NR groups showed significantly greater arthrogenic contracture than the C group (P&#8201;&lt;&#8201;0.05). Compared to the NR group, the EI and CI groups exhibited significant improvement in arthrogenic contracture (P&#8201;&lt;&#8201;0.05). Conversely, the EI group showed lower contracture than the CI group (P&#8201;&lt;&#8201;0.05). Similar results were observed for collagen deposition and the protein levels of collagens I and III. The intervention groups (EI and CI groups) showed higher levels of p-Nrf2/Nrf2 and HO-1 than the NR group (P&#8201;&lt;&#8201;0.05). Moreover, the EI group exhibited higher levels of p-PKA/PKA, p-Nrf2/Nrf2, and HO-1 than the CI group (P&#8201;&lt;&#8201;0.05). However, no significant difference was found in the A₂AR levels among the five groups (P&#8201;&gt;&#8201;0.05). ESWT may activate A₂AR, leading to the phosphorylation of PKA. Subsequently, Nrf2 may be activated, resulting in the upregulation of HO-1, which then reduces collagen deposition and alleviates immobilization-induced joint capsule fibrosis.

<b>Objective:</b> To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1&#945;/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. <b>Methods:</b> Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1&#945;/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1&#945; in ccRCC cell lines. HeLa cells were treated with CoCl₂ and hypoxia (1%O₂) to activate HIF-1&#945; and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1&#945;-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1&#945;/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. <b>Results:</b> Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1&#945; expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1&#945; in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1&#945; in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1&#945; in Caki-2 cells inhibited the mRNA and protein levels of ACLY (<i>P</i>&lt;0.001 for all). CoCl₂ and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1&#945; (<i>P</i>&lt;0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1&#945; could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (<i>P</i>&lt;0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1&#945; and ACLY. The depletion of HIF-1&#945; could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1&#945; knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (<i>P</i>&lt;0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (<i>P</i>&lt;0.001). <b>Conclusions:</b> VHL mutation-mediated HIF-1&#945; overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1&#945;/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.

Addressing spinal cord injury (SCI) through stem cell therapy is currently at the forefront of medical research despite its complexity. In this study, we investigated the potential of the Noggin protein in promoting the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neuronal cells. We transplanted induced cells into a rat model with spinal cord injury. This exploration proposes an innovative perspective on stem cell therapies for spinal cord injuries.

Approximately 50% of hepatocellular carcinoma (HCC) arises due to the infection by hepatitis B virus X protein (HBx). Sorafenib, a unique targeted oral kinase inhibitor, is the therapeutic agent of choice for advanced HCC. The mechanism of HBx in drug resistance of sorafenib-resistant HCC cells was evaluated in this study.

Chemodynamic therapy (CDT) has witnessed significant advancements in recent years due to its specific properties. Its association with photodynamic therapy (PDT) has also garnered increased attention due to its mutually reinforcing effects. However, achieving further enhancement of the CDT/PDT efficacy remains a major challenge. In this study, we have developed an integrated nanosystem comprising a Fenton catalyst and multifunctional photosensitizers to achieve triply enhanced CDT/PDT through photothermal effects, H₂O₂ elevation, and GSH consumption. We prepared nano-ZIF-8 vesicles as carriers to encapsulate ferrocene-(phenylboronic acid pinacol ester) conjugates (Fc-BE) and photosensitizers IR825. Subsequently, cinnamaldehyde-modified hyaluronic acid (HA-CA) was coated onto ZIF-8 through metal coordination interactions, resulting in the formation of active targeting nanoparticles (NPs@Fc-BE&amp;IR825). Upon cellular internalization mediated by CD44 receptors, HA-CA elevated H₂O₂ levels, while released Fc-BE consumed GSH and catalyzed H₂O₂ to generate highly cytotoxic hydroxyl radicals (&#183;OH). Furthermore, NIR irradiation led to increased &#183;OH production and the generation of singlet oxygen (¹O₂), accompanied by a greater GSH consumption. This accelerated and strengthened amplification of oxidative stress can be harnessed to develop highly effective CDT/PDT nanoagents.

BACKGROUND A non-infectious inflammatory reaction against replaced aortic graft for aortic dissection often manifests as fever, malaise, and peri-graft effusion. It usually lasts less than 1 month and subsides spontaneously without immunosuppressive treatment. CASE REPORT A 49-year-old man underwent ascending aorta and total arch replacement for acute thoracic aortic dissection. He had fever, malaise, nausea, and elevated serum C-reactive protein for 1 month postoperatively. Pathological examination of the aorta revealed no aortitis, and repeated blood cultures were negative. We also noted periaortic graft fluid collection, and a small amount of pleural and pericardial effusions. We suspected post-pericardiotomy syndrome. Colchicine and prednisolone were administered, with an excellent clinical response. Three weeks after discontinuation of a 7-week prednisolone treatment, the same symptoms recurred and gradually worsened. Prednisolone was restarted 6 months after the first surgery, with good clinical response. Thereafter, he developed left-sided weakness and dysarthria, being diagnosed as ischemic stroke. Contrast-enhanced computed tomography revealed fluid collection with contrast leak around the aortic grafts, suggesting peel dehiscence, and thrombus formation in anastomotic pseudoaneurysm. He underwent surgical repair. He was diagnosed with non-infectious periaortitis, likely due to an immune reaction to the grafts, based on an excellent clinical response to immunosuppressive therapy. CONCLUSIONS We report a case of non-infectious periaortitis around a thoracic aortic graft, probably with an immune-mediated mechanism, requiring immunosuppressive treatment. When fever persists after aortic graft replacement surgery, non-infectious periaortitis should be considered and immunosuppressive treatment should be considered to prevent critical complications of anastomotic pseudoaneurysm and graft dehiscence.

The worldwide appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated significant concern and posed a considerable challenge to global health. Phosphorylation is a common post-translational modification that affects many vital cellular functions and is closely associated with SARS-CoV-2 infection. Precise identification of phosphorylation sites could provide more in-depth insight into the processes underlying SARS-CoV-2 infection and help alleviate the continuing COVID-19 crisis. Currently, available computational tools for predicting these sites lack accuracy and effectiveness. In this study, we designed an innovative meta-learning model, Meta-Learning for Serine/Threonine Phosphorylation (MeL-STPhos), to precisely identify protein phosphorylation sites. We initially performed a comprehensive assessment of 29 unique sequence-derived features, establishing prediction models for each using 14 renowned machine learning methods, ranging from traditional classifiers to advanced deep learning algorithms. We then selected the most effective model for each feature by integrating the predicted values. Rigorous feature selection strategies were employed to identify the optimal base models and classifier(s) for each cell-specific dataset. To the best of our knowledge, this is the first study to report two cell-specific models and a generic model for phosphorylation site prediction by utilizing an extensive range of sequence-derived features and machine learning algorithms. Extensive cross-validation and independent testing revealed that MeL-STPhos surpasses existing state-of-the-art tools for phosphorylation site prediction. We also developed a publicly accessible platform at https://balalab-skku.org/MeL-STPhos. We believe that MeL-STPhos will serve as a valuable tool for accelerating the discovery of serine/threonine phosphorylation sites and elucidating their role in post-translational regulation.

Efficient plant acclimation to changing environmental conditions relies on fast adjustments of the transcriptome, proteome, and metabolome. Regulation of enzyme activity depends on the activity of specific chaperones, chemical post-translational modifications (PTMs) of amino acid residues, and changes in the cellular and organellar microenvironment. Central to carbon assimilation, and thus plant growth and yield, Rubisco activity is regulated by its chaperone Rubisco activase (Rca) and by adjustments in the chloroplast stroma environment. Focused on crops, this review highlights the main PTMs and stromal ions and metabolites affecting Rubisco and Rca in response to environmental stimuli. Rca isoforms differ in regulatory properties and heat sensitivity, with expression changing according to the surrounding environment. Much of the physiological relevance of Rubisco and Rca PTMs is still poorly understood, though some PTMs have been associated with Rubisco regulation in response to stress. Ion and metabolite concentrations in the chloroplast change in response to variations in light and temperature. Some of these changes promote Rubisco activation while others inhibit activation, deactivate the enzyme, or change the rates of catalysis. Understanding these regulatory mechanisms will aid the development of strategies to improve carbon fixation by Rubisco under rapidly changing environments as experienced by crop plants.

Galectin-1 (Gal-1), a member of a highly conserved family of animal lectins, plays a crucial role in controlling inflammation and neovascularization. However, the potential role of Gal-1 in preventing myocarditis remains uncertain. We aimed to explore the functions and mechanisms of Gal-1 in preventing myocarditis. In vivo, C57/BL6 mice were pre-treated with or without Gal-1 and then exposed to lipopolysaccharide (LPS) to induce myocarditis. Subsequently, cardiac function, histopathology, inflammation, oxidative stress, and apoptosis of myocardial tissues were detected. Following this, qRT-PCR and Western blotting were applied to measure iNOS, COX2, TXNIP, NLRP3 and Caspase-1 p10 expressions. In vitro, H9c2 cells pre-treated with different doses of Gal-1 were stimulated by LPS to induce myocarditis models. CCK8, flow cytometry and reactive oxygen species (ROS) assay were then employed to estimate cell viability, apoptosis and oxidative stress. Furthermore, Nrf2 and HO-1 protein expressions were evaluated by Western blotting in vivo and in vitro. The results showed that in vivo, Gal-1 pre-treatment not only moderately improved cardiac function and cardiomyocyte apoptosis, but also ameliorated myocardial inflammation and oxidative damage in mice with myocarditis. Furthermore, Gal-1 inhibited TXNIP-NLRP3 inflammasome activation. In vitro, Gal-1 pre-treatment prevented LPS-induced apoptosis, cell viability decrease and ROS generation. Notably, Gal-1 elevated HO-1, total Nrf2 and nuclear Nrf2 protein expressions both in vivo and in vitro. In conclusion, pre-treatment with Gal-1 exhibited cardioprotective effects in myocarditis via anti-inflammatory and antioxidant functions, and the mechanism may relate to the Nrf2 pathway, which offered new solid evidence for the use of Gal-1 in preventing myocarditis.

This research delves into the implications of the RNA binding motif, single stranded interacting protein 2 (RBMS2)-a gene associated with tumor-suppressing functions-in the context of kidney renal clear cell carcinoma (ccRCC). Through meticulous exploration of online databases, we have identified a negative association between RBMS2 expression and adverse clinico-pathological features, such as advanced TNM stage. Furthermore, our findings indicate that RBMS2 acts as a prognostic predictor for clinical outcomes in ccRCC, evidenced by both univariate and multivariate analyses. Cellular assays have corroborated these findings, revealing that an overexpression of RBMS2 curtails ccRCC cell proliferation and migration. Additionally, our research has unearthed links between RBMS2 and immune infiltration within the ccRCC tumor microenvironment. Collectively, our results underscore the tumor-inhibiting role of RBMS2 in ccRCC and spotlight its potential as a prognostic marker and therapeutic intervention target.

Label="Introduction" NlmCategory="UNASSIGNED">Research shows the correlation between angiotensin-converting enzyme (ACE) deletion and insertion (D/I) polymorphism and COVID-19 risk; yet, conclusive evidence is still lacking. Thus, a meta-analysis of relevant articles was performed to more accurately estimate the relationship of ACE I/D polymorphism with the risk of COVID-19. <i>Material and Methods</i>. Relevant literature from the PubMed database was systematically reviewed, and odds ratios (ORs) and associated 95% confidence intervals (CIs) were measured. Additionally, the metapackage from Stata version 15.0 was used for statistical analysis.

The authors requested retraction after reviewing the manuscript and determining that there were errors in the data. Reference: Yanli Hong, Yanyun Yin, Yong Tan, Ke Hong, Huifang Zhou. The Flavanone, Naringenin, Modifies Antioxidant and Steroidogenic Enzyme Activity in a Rat Model of Letrozole-Induced Polycystic Ovary Syndrome. Med Sci Monit, 2019; 25: 395-401. DOI: 10.12659/MSM.912341.

This study evaluated the effect of fibroblast growth factor receptor 3 (FGFR3) on damaged hypertrophic chondrocytes of Kashin-Beck disease (KBD). Immunohistochemical staining was used to evaluate FGFR3 expression in growth plates from KBD rat models and engineered cartilage. In vitro study, hypertrophic chondrocytes were pretreated by FGFR3 binding inhibitor (BGJ398) for 24 h before incubation at different T-2 toxin concentrations. Differentiation -related genes (Runx2, Sox9, and Col Ⅹ) and ECM degradation -related genes (MMP-13, Col Ⅱ) in the hypertrophic chondrocytes were analyzed using RT-PCR, and the corresponding proteins were analyzed using western blotting. Hypertrophic chondrocytes death was detected by the Annexin V/PI double staining assay. The integrated optical density of FGFR3 staining was increased in knee cartilage of rats and engineered cartilage treated with T-2 toxin. Both protein and mRNA levels of Runx2, Sox9, Col Ⅱ, and Col Ⅹ were decreased in a dose-dependent manner when exposed to the T-2 toxin and significantly upregulated by 1 μM BGJ398. The expression of MMP-1, MMP-9, and MMP-13 increased in a dose-dependent manner when exposed to T-2 toxin and significantly reduced by 1 μM BGJ398. 1 μM BGJ398 could prevent early apoptosis and necrosis induced by the T-2 toxin. Inhibiting the FGFR3 signal could alleviate extracellular matrix degradation, abnormal chondrocytes differentiation, and excessive cell death in T-2 toxin-induced hypertrophic chondrocytes.

Being an underdiagnosed and under or insufficiently treated condition, surgical pancreatic exocrine insufficiency (PSP) is the condition in which pancreatic enzymes are insufficient for digestion because of gastrointestinal (GI) surgery involving the upper GI tract, biliary ducts, or the pancreas, and and leading to potential malnutrition and deterioration in quality of life. Age, obesity, history of tobacco use, family history of diabetes, surgery due to a malignant tumor, presence of steatorrhea, jaundice, weight loss, and intraoperative findings of hard pancreatic texture have been associated with a higher risk of PSP. Pancreatoduodectomy (PD) has demonstrated an increased risk of developing PSP, with a prevalence between 19-100%. Distal pancreatectomy (DP) and central pancreatectomy (CenP) are associated with less risk of PSP, with a prevalence of 0-82% and 3.66-8.7%, respectively. In patients with chronic pancreatitis (CP), PSP was associated with 80% in Partington-Rochelle procedure, 86% in Frey procedure, 80% in duodenum preserving pancreatic head procedure, >60% in PD and 27.5-63% in DP. Fecal elastase-1 (FE-1) is a generally accepted tool for diagnosis. Treatment is recommended to start as soon as a diagnosis is achieved, or clinical suspicion is high. Pancreatic enzyme replacement therapy improves symptoms of malabsorption, facilitates weight gain, and ultimately improves patients' quality of life. Starting dosage is between 10,000-50,000 units in snacks and 50,000-75,000 units in main meals, administered throughout food intake, though further data specifically on PSP are needed. Follow-up in PSP is recommended on an on-demand basis, where malnutrition should be assessed.

Cardiac fibrosis (CF) in response to persistent exogenous stimuli or myocardial injury results in cardiovascular diseases (CVDs). Protein tyrosine phosphatase 1B (PTP1B) can promote collagen deposition through regulating AMPK/TGF-β/Smads signaling pathway, and PTP1B knockout improves cardiac dysfunction against overload-induced heart failure. Oleanolic acid (OA) has been proven to be an inhibitor of PTP1B, and its anti-cardiac remodeling effects have been validated in different mouse models. To improve the bioactivity of OA and to clarify whether OA derivatives with stronger inhibition of PTP1B activity have greater prevention of cardiac remodeling than OA, four new OA derivatives were synthesized and among them, we found that compound B had better effects than OA in inhibiting cardiac fibrosis both in vivo in the isoproterenol (ISO)-induced mouse cardiac fibrosis and in vitro in the TGF-β/ISO-induced 3T3 cells. Combining with the results of molecular docking, surface plasmon resonance and PTP1B activity assay, we reported that OA and compound B directly bound to PTP1B and inhibited its activity, and that compound B showed comparable binding capability but stronger inhibitory effect on PTP1B activity than OA. Moreover, compound B presented much greater effects on AMPK activation and TGF-β/Smads inhibition than OA. Taken together, OA derivative compound B more significantly alleviated cardiac fibrosis than OA through much greater inhibition of PTP1B activity and thus much stronger regulation of AMPK/TGF-β/Smads signaling pathway.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

The incidence of breast cancer in women has increased year by year, becoming one of the most common malignant tumors in females worldwide. Most patients can be treated with surgery and endocrine drugs, but there are still some patients who lack effective treatment, such as triple-negative breast cancer (TNBC). Nectin-4, a protein encoded by poliovirus receptor-associated protein 4, is a Ca²⁺-independent immunoglobulin-like protein. It is mainly involved in the adhesion between cells. In recent years, studies have found that Nectin-4 is overexpressed in breast cancer and several other malignancies. Otherwise, several monoclonal antibodies and inhibitors targeting Nectin-4 have shown prosperous outcomes, so Nectin-4 has great potential to be a therapeutic target for breast cancer. The present review systematically describes the significance of Nectin-4 in each aspect of breast cancer, as well as the molecular mechanisms of these aspects mediated by Nectin-4. We further highlight ongoing or proposed therapeutic strategies for breast cancer specific to Nectin-4.

Lateral organ boundaries (LOB) domain (<i>LBD</i>) genes, a gene family that encodes the transcription factors (TFs) of plants, plays crucial functions in the development and growth of plants. Currently, genome-wide studies of the <i>LBD</i> family are still limited to tomato (<i>Solanum lycopersicum</i> L.), which is considered an important economic crop. In this study, we performed a genome-wide analysis of <i>LBD</i> in tomato. In total, 56 <i>LBDs</i> were found in the tomato genome. Protein alignment and phylogenetic classification showed that <i>LBDs</i> were conserved with other species. Since light emitting diodes (LEDs) light have promising applications for tomato growth. To better understand the potential function of <i>LBDs</i> in response to LED light in tomato, we conducted a genome-wide expression analysis of <i>LBD</i> genes under different light conditions. As expected, different LED lights affected the tomato growth (e.g. hypocotyl length). RNA-seq data showed that eight <i>LBDs</i> in tomato seedlings were differentially expressed under different light treatments, including white, blue, red, and far-red light, compared to the dark-grown condition. It indicates that these <i>LBDs</i> might regulate plant development in different LED light conditions. Interestingly, two <i>LBD</i> genes (<i>SlLBD1</i> and <i>SlLBD2</i>) were found to be differentially expressed in four distinct lights, which might be involved in regulating the plant architecture via a complicated TF network, which can be taken into consideration in further investigation.

<i>Discoviridae</i> is a family of negative-sense RNA viruses with genomes of 6.2-9.7&#8201;kb that have been associated with fungi and stramenopiles. The discovirid genome consists of three monocistronic RNA segments with open reading frames (ORFs) that encode a nucleoprotein (NP), a nonstructural protein (Ns), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family <i>Discoviridae</i>, which is available at ictv.global/report/discoviridae.

Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.

Polycyclic aromatic hydrocarbons (PAHs) are genotoxic, carcinogenic, and persistent in the environment and are therefore of great concern in the environmental protection field. Due to the inherent recalcitrance, persistence and nonreactivity of PAHs, they are difficult to remediate via traditional water treatment methods. In recent years, microbial remediation has been widely used as an economical and environmentally friendly degradation technology for the treatment of PAH-contaminated water. Various bacterial and microalgal strains are capable of potentially degrading or transforming PAHs through intrinsic metabolic pathways. However, their biodegradation potential is limited by the cytotoxic effects of petroleum hydrocarbons, unfavourable environmental conditions, and biometabolic limitations. To address this limitation, microbial communities, biochemical pathways, enzyme systems, gene organization, and genetic regulation related to PAH degradation have been intensively investigated. The advantages of algal-bacterial cocultivation have been explored, and the limitations of PAHs degradation by monocultures of algae or bacteria have been overcome by algal-bacterial interactions. Therefore, a new model consisting of a "microalgal-bacterial consortium" is becoming a new management strategy for the effective degradation and removal of PAHs. This review first describes PAH pollution control technologies (physical remediation, chemical remediation, bioremediation, etc.) and proposes an algal-bacterial symbiotic system for the degradation of PAHs by analysing the advantages, disadvantages, and PAH degradation performance in this system to fill existing research gaps. Additionally, an algal-bacterial system is systematically developed, and the effects of environmental conditions are explored to optimize the degradation process and improve its technical feasibility. The aim of this paper is to provide readers with an effective green and sustainable remediation technology for removing PAHs from aquatic environments.

In understanding stress response mechanisms in fungi, cold stress has received less attention than heat stress. However, cold stress has shown its importance in various research fields. The following study examined the cold stress response of six Pseudogymnoascus spp. isolated from various biogeographical regions through a proteomic approach. In total, 2541 proteins were identified with high confidence. Gene Ontology enrichment analysis showed diversity in the cold stress response pathways for all six Pseudogymnoascus spp. isolates, with metabolic and translation-related processes being prominent in most isolates. 25.6% of the proteins with an increase in relative abundance were increased by more than 3.0-fold. There was no link between the geographical origin of the isolates and the cold stress response of Pseudogymnoascus spp. However, one Antarctic isolate, sp3, showed a distinctive cold stress response profile involving increased flavin/riboflavin biosynthesis and methane metabolism. This Antarctic isolate (sp3) was also the only one that showed decreased phospholipid metabolism in cold stress conditions. This work will improve our understanding of the mechanisms of cold stress response and adaptation in psychrotolerant soil microfungi, with specific attention to the fungal genus Pseudogymnoascus.

To evaluate health-related quality of life (HRQoL) of people with a high-risk skin melanoma after completion of the primary surgical treatment over time, as well as, to identify factors associated with better HRQoL at the beginning and at the end of follow-up. The study included subjects with histopathologically confirmed high-risk skin melanoma in clinical stages IIC, IIIA, IIIB, and IIIC, in whom clinical and radiographic signs of the disease were not confirmed after primary surgical treatment. The HRQoL was evaluated using Short Form-36 (SF-36) after completion of primary surgical treatment (start of follow-up) and after 6 to 12 months (end of follow-up). A total of 71 people completed SF-36 at both points in time. There were no significant differences between the initial and the follow-up total HRQoL score (t = 1.118; p = 0.267). At the start of follow-up, having fewer depressive symptoms, better functional status and lower vitamin D serum levels were associated with a better total HRQoL score. At the end of follow-up, having lower Breslow depth and being employed at the start of follow-up, having fewer depressive symptoms and lower C-reactive protein (CRP) serum levels at follow-up, and not developing metastases over follow-up were associated with a higher total HRQoL scores. The HRQoL of people with high-risk melanoma did not change in the year following the complete removal of the tumor. However, presence of depressive symptoms and metastases seem to have the strongest impact on poorer quality of life after surgery.

Tyramides are produced in microgram quantities by males of species in the large Myrmicine ant sub-family (> 7000 species). Tyramides are transferred to female sexuals during mating where a specific female sexual evolved enzyme hydrolyzes the tyramides to the biogenic amine, tyramine. Tyramine is a ligand for receptors that rapidly activate reproductive development in the newly mated queen-previously reproductively inhibited by the mother queen. Without this elaborate biogenic amine precursor and co-evolved female sexual derived tyramide hydrolase, the defenseless newly mated queen's worker production would be delayed by up to 6 days, which could be lethal to the new queen. This is one of possibly several ant species separation mechanisms evolved to maintain species integrity. Here we report two methyl-branched tyramides from harvester ant, Pogonomyrmex badius, males, including one highly branched tyramide not previously reported.

Inborn errors of metabolism (IEM) are a group of about 500 rare genetic diseases with large diversity and complexity due to number of metabolic pathways involved in. Establishing a correct diagnosis and identifying the specific clinical phenotype is consequently a difficult task. However, an inclusive diagnosis able in capturing the different clinical phenotypes is mandatory for successful treatment. However, in contrast with Garrod's basic assumption "one-gene one-disease," no "simple" correlation between genotype-phenotype can be vindicated in IEMs. An illustrative example of IEM is Phenylketonuria (PKU), an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism, ascribed to variants of the phenylalanine hydroxylase (PAH) gene encoding for the enzyme complex phenylalanine-hydroxylase. Blood values of Phe allow classifying PKU into different clinical phenotypes, albeit the participation of other genetic/biochemical pathways in the pathogenetic mechanisms remains elusive. Indeed, it has been shown that the most serious complications, such as cognitive impairment, are not only related to the gene dysfunction but also to the patient's background and the participation of several nongenetic factors.Therefore, a Systems Biology-based strategy is required in addressing IEM complexity, and in identifying the interplay between different pathways in shaping the clinical phenotype. Such an approach should entail the concerted investigation of genomic, transcriptomics, proteomics, metabolomics profiles altogether with phenylalanine and amino acids metabolism. Noticeably, this "omic" perspective could be instrumental in planning personalized treatment, tailored accordingly to the disease profile and prognosis.